U.S. patent application number 09/826561 was filed with the patent office on 2002-01-17 for optical components for microarray analysis.
Invention is credited to Brown, Carl S., Goodwin, Paul C., Quarre, Steven C., Reese, Steven A..
Application Number | 20020005493 09/826561 |
Document ID | / |
Family ID | 26890173 |
Filed Date | 2002-01-17 |
United States Patent
Application |
20020005493 |
Kind Code |
A1 |
Reese, Steven A. ; et
al. |
January 17, 2002 |
Optical components for microarray analysis
Abstract
The method of illumination of a microarray sample may contribute
to the signal-to-background ratio. An oblique illumination
technique is used to reduce the reflections from the sample to the
detector. The sample may also be moved to the backside of the
sample support to reduce the reflections caused by the sample
support. In addition, a parallel scanning technique may be used to
ensure proper alignment of the sample.
Inventors: |
Reese, Steven A.;
(Shoreline, WA) ; Quarre, Steven C.; (Woodinville,
WA) ; Brown, Carl S.; (Seattle, WA) ; Goodwin,
Paul C.; (Shoreline, WA) |
Correspondence
Address: |
JAMES T. HAGLER
Fish & Richardson P.C.
Suite 500
4350 La Jolla Village Drive
San Diego
CA
92122
US
|
Family ID: |
26890173 |
Appl. No.: |
09/826561 |
Filed: |
April 4, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60194574 |
Apr 4, 2000 |
|
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Current U.S.
Class: |
250/459.1 |
Current CPC
Class: |
G01N 21/6458 20130101;
G01N 2021/6484 20130101; G01N 21/6452 20130101; G01N 2021/6471
20130101 |
Class at
Publication: |
250/459.1 |
International
Class: |
G01N 021/64 |
Claims
What is claimed is:
1. A method of illuminating a sample comprising: positioning the
sample beneath a detector; and directing a light source at the
sample at an angle such that reflections of the light source off
the sample are directed away from the detector.
2. The method of claim 1, further comprising setting the angle to
an oblique angle.
3. The method of claim 1, further comprising setting the angle so
that the reflections are directed away from the sample at
approximately the angle.
4. The method of claim 1, further comprising setting the angle
outside an acceptance angle of an objective lens.
5. The method of claim 1, wherein the sample is a microarray
sample.
6. The method of claim 1, further comprising providing illumination
using fiber optics.
7. The method of claim 1, further comprising collecting
fluorescence generated at the sample with the detector.
8. A method of illuminating a sample comprising: positioning the
sample on a lower side of a sample support; and directing an
illumination source through the sample support to the sample.
9. The method of claim 8, further comprising directing the
illumination source at the sample at an oblique angle.
10. The method of claim 8, further comprising providing
illumination using fiber optics.
11. The method of claim 8, further comprising collecting
fluorescence generated at the sample with a detector.
12. The method of claim 11, further comprising positioning the
sample support between the sample and the detector.
13. The method of claim 8, wherein the illumination source refracts
through the sample support.
14. The method of claim 8, further comprising positioning a
microarray sample on the sample support.
15. A method of obtaining a plurality of samples of a microarray
comprising: exciting the microarray with an illumination source;
aligning a first portion of the microarray with a detector;
collecting the fluorescence from the first portion of the
microarray; moving the microarray to align a second portion of the
microarray with the detector; and collecting the fluorescence from
the second portion of the microarray.
16. The method of claim 15, further comprising repositioning the
microarray until fluorescence is obtained from the entire
microarray.
17. The method of claim 15, further comprising collecting the
fluorescence of each subsequent portion of the microarray prior to
further repositioning.
18. The method of claim 15, further comprising adjusting a
fluorescence channel of the illumination source.
19. The method of claim 18, further comprising changing
interference filters to adjust the fluorescence channel.
20. The method of claim 15, further comprising collecting the
fluorescence with a charge-coupled device.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/194,574, filed Apr. 4, 2000.
TECHNICAL FIELD
[0002] This invention relates to microarray analysis, and more
particularly to optical components used in microarray analysis.
BACKGROUND
[0003] Biomedical research has made rapid progress based on
sequential processing of biological samples. Sequential processing
techniques have resulted in important discoveries in a variety of
biologically related fields, including, among others, genetics,
biochemistry, immunology and enzymology. Historically, sequential
processing involved the study of one or two biologically relevant
molecules at the same time. These original sequential processing
methods, however, were quite slow and tedious. Study of the
required number of samples (up to tens of thousands) was time
consuming and costly.
[0004] A breakthrough in the sequential processing of biological
specimens occurred with the development of techniques of parallel
processing of the biological specimens, using fluorescent marking.
A plurality of samples are arranged in arrays, referred to herein
as microarrays, of rows and columns into a field, on a substrate
slide or similar member. The specimens on the slide are then
biochemically processed in parallel. The specimen molecules are
fluorescently marked as a result of interaction between the
specimen molecule and other biological material. Such techniques
enable the processing of a large number of specimens very
quickly.
[0005] In microarray experiments, the sample volume may be very
limited. Furthermore, amplification methods (e.g. polymerase chain
reaction, etc.) may not be sufficiently quantitative for this
application. Even more so, the very biomolecular species that are
most likely to prove to be important in these assays are the very
ones that are least abundant. All of these factors influence the
need for a microarray scanner to be as sensitive as possible. For a
fluorescent application such as this, one critical decision is how
to deliver as much excitation light as possible without increasing
the background of the image. To do otherwise has no value since the
signal-to-background ratio would not improve.
SUMMARY
[0006] The method of illumination of a microarray sample may
contribute to the signal-to-background ratio. An oblique
illumination technique is used to reduce the reflections from the
sample to the detector. The sample may also be moved to the
backside of the sample support to reduce the reflections caused by
the sample support. In addition, a parallel scanning technique may
be used to ensure proper alignment of the sample.
DESCRIPTION OF DRAWINGS
[0007] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
[0008] FIG. 1 is a front view of an illumination system using a
beam splitter as is known in the art.
[0009] FIG. 2 is a front view of an illumination system using an
oblique illumination light path according to one embodiment of the
present invention.
[0010] FIG. 3 is a front view of an illumination system using
front-side illumination showing the light propagation according to
one embodiment of the present invention.
[0011] FIG. 4 is a front view of an illumination system using
back-side illumination showing the light propagation according to
one embodiment of the present invention.
[0012] FIG. 5 illustrates a parallel scanning technique to obtain
samples during microarray analysis according to one embodiment of
the present invention.
[0013] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0014] The most common method of illuminating the sample for
fluorescence is to use so called epi-illumination as illustrated in
FIG. 1. In this method, the illumination and the emission share at
least part of the optical train. Light enters the optic train from
a source 105 and reflects off of a beam splitter 110. The light
then enters an objective 115, travels through a series of internal
lenses 120, and on to the sample 130. The sample 130 is typically
mounted on a support 125, such as a glass microscope slide.
Fluorescent light 135 that is generated at the sample traverses
back through the objective lens 120 and the beam splitter 110 and
continues on for data collection. The sensitivity of
epi-illumination based systems is limited by the autofluoresence of
the optical elements and reflection of illumination light off of
the sample 130 and the internal lens elements 120 which contribute
to background in the collected image. The signal in an
epi-illumination system is further limited by the efficiency with
which the beam splitter 110 can transmit and reflect light. The
beam splitter 110 also greatly reduces the flexibility of the
system since the beam splitter 110 must be matched to the
excitation and emission filters.
[0015] One embodiment of the present invention uses oblique
illumination for microarrays as seen in FIG. 2. With oblique
illumination, light is delivered through fiber optic fibers 205 or
some other comparable light source outside of the objective lens
115. The illumination is directed at an angle 210 such that the
illumination is outside of the acceptance angle of the objective
lens 115. In one example, the light is delivered at a 45.degree.
angle, well outside of the 11.5.degree. angle of an 4.times./0.2NA
objective lens. Any fluorescence generated at the sample 130 is
collected by the objective lens 115. The portion of the
illumination light that is reflected 220 by the sample is deflected
at the illumination angle 210, in this example, 45 degrees 225. In
so doing, neither the illumination nor the reflection 220 of the
illumination are collected by the objective lens 115 as they fall
outside of the acceptance angle of the lens. As the illumination
did not traverse any of the light collection optics, there is no
background generated by either internal reflections in the
objective lens 115 or by autofluorescence of the optical
components. The net effect is bright illumination to the sample
with greatly reduced contributions to the background which
generates superior signal-to-background over conventional
epi-illumination methods.
[0016] In addition to the light path, the orientation of the
specimen also effects the illumination. With front-side
illumination and detection, the sample 130 is closest to the optics
as seen in FIG. 3. In front-side illumination and detection, the
sample 130 sits on the top side of the sample support 125. The
illumination source 205 and the objective 115 are on the same side
of the sample support 125 as the sample 130. Fluorescence is
generated at the sample 130 and a portion of the fluorescence 315
is collected directly by the objective 115 and transmitted on to
the detector. Of all of the fluorescence generated at the sample
130, a portion 305 enters the sample support 125 and internally
reflects back 310 past the sample 130 and is collected by the
objective 115. This internal reflection 310 contributes undesirably
to the total fluorescence in the form of background. As a result,
the signal-to-background ratio is significantly reduced.
[0017] To reduce this reflection and increase the
signal-to-background ratio, the sample support 135 is inverted
creating Back-Side Illumination and Detection as seen in FIG. 4.
With Back-Side Illumination and Detection, the sample 130 is on the
opposite side of the sample support 125 than the objective 115 and
source illumination 205. Light 405 from the source 205 refracts
through the sample support 125 and illuminates the sample 130.
Fluorescence 410 generated by the sample 130 transmits through the
sample support 125, and a portion 407 travels into the objective
115 and on to the detector. Light internally reflected 415 by the
support 125 is directed away from the detector. Some small number
of photons may reflect an additional time 420 and make it to the
detector, but the number of these secondary reflections relative to
the total fluorescent signal is small. The total amount of signal
using Back-Side Illumination and Detection is nearly twice what it
is for Front-Side Illumination
[0018] Most applications for microarray scanners use internal
controls for every sample. That is, for every measurement made,
there is an independent control sample. The experimental value is
then expressed as a ratio of the experimental value normalized to
the control value. This is referred to as a ratiometric
measurement. Ratiometric measurements are powerful methods in that
every sample is independently controlled. The weakness of
ratiometric measurements is that they place strict requirements on
the instrumentation that generates the measurements. Division, the
mathematical operation that is used for generating ratios, does not
gracefully tolerate values that approach zero. This effect is
primarily seen as the denominator intensity approaches zero. I that
case, this drives the ratio to infinity and values of zero become
undefined. Consequently, in imaging applications, exact alignment
of images representing the experimental and control signals are
critical. In commercially available laser scanning instruments, one
of two methods for acquiring multiple wavelength images in
employed. In some systems, the sample is scanned once for each
fluorochrome in the sample. Since the different scans require a
different mechanical scanning of the sample, the images are very
difficult to perfectly align. In other systems, multiple
fluorochromes are scanned for at the same time using off-set points
for each wavelength. Even in this method, the images are often
misaligned. In the present invention, the optical path is held
constant and the sample is scanned beneath the optics. At each
physical location, all of the fluorochromes in use are acquired in
succession (FIG. 5). Consequently, the images from the acquisitions
of each fluorochrome are limited not by mechanical rescanning but
solely by the chromatic error in the optics. By controlling the
chromatic error (through careful lens design) the chromatic error
for each point in the image is smaller than the size of our
detection element (i.e. sub-pixel) so it will not deteriorate the
ratiometric data.
[0019] FIG. 5 illustrate a Parallel Scanning technique used in the
present invention. With Parallel Scanning, light is generated by a
single source such as an arc lamp 505 that is broad spectrum. An
interference filter 510 is used to select excitation wavelengths.
The light is launched into a fiber bundle 515 that delivers light
essentially uniformly to a panel 520 on the sample 525.
Fluorescence is collected by optics, such as an objective lens 530
and passes through an additional interference filter 535 which is
used to achieve a high level of wavelength specificity. The light
is then detected by a parallel collection device such as a
charge-coupled device (CCD) camera 540. In order to acquire
additional fluorescence channels, only the interference filters
510, 535 are changed and the remainder of the opto-mechanical path
is held fixed. The interference filters 510, 535 may be held in a
housing of sealed filter wheels (not shown). The filter wheels may
include mechanical and sensor technology to easily change the
current filter. To scan the remainder of the sample 525, the sample
525 is moved panel by panel under the fixed optical path until the
entire sample 525 has been scanned. In this way, the alignment of
the images representing each fluorescent probe are in alignment to
greater precision than the size of the individual detectors in the
CCD camera 540.
[0020] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *